Milk protein isolate and method for preparing same

ABSTRACT

The invention relates to a novel process for isolating milk proteins from milk or from whey; its subject is also a milk protein fraction obtained by this process and its use for the preparation of pharmaceutical or food compositions.

The subject of the present invention is a novel process for isolatingmilk proteins, the composition, milk protein isolate or milk proteinfraction, resulting therefrom and its applications, in particular itsfood and pharmaceutical applications.

Several prior art documents describing the purification of milk proteinsare known.

The document EP-253395 describes the production of a bovine lactoferrinof high purity (>80% in a single step or >98% in two steps) byadsorption of milk or of whey onto a cation-exchange resin comprisingcarboxymethyl radicals, followed by rinsing and desorption of thelactoferrin.

The document EP-348508 also describes a process for preparing a bovinelactoferrin of high purity (>95%) by adsorption of milk or whey onto acation-exchange resin of the polysaccharide type comprising sulfuricacid ester functional groups and elution with an aqueous salt solution.

The document EP-298875 describes a process which makes it possible toisolate certain whey proteins by adsorption onto a porous mineralsupport in the form of grains, these grains being coated with anamine-containing polysaccharide layer having at the surface acidfunctional groups such as carboxylic or sulfonic groups.

The document EP-418704 describes a process for sequentially purifyinglactoferrin and lactoperoxidase by elution on a column loaded with apolysaccharide resin grafted by sulfonic acid groups.

The document U.S. Pat. No. 6,096,870 describes a process for thesequential separation of whey proteins by elution on cationic resins.

The document EP-1017286 describes a process for the sequentialseparation of whey proteins by radial flow chromatography.

Lactoferrin and lactoperoxidase are milk proteins with advantageousproperties, their capacity to bind iron confers on them a role ofantibacterial agent against bacteria whose metabolism requires largequantities of iron. Lactoperoxidase is essential for labeling proteinwith iodine. Lactoferrin promotes the growth of lymphocytes and promotesthe absorption of iron by the body; it regulates the differentiation ofleukocytes and it inhibits the peroxidation of lipids.

However, all these documents relate to processes aimed at a lactoferrinand/or a lactoperoxidase which are as pure as possible, that is to sayprocesses aimed at reducing as much as possible the proportion of theother proteins initially present in milk or whey. In addition, theconditions for binding the raw material and for eluting the proteins aregentle conditions, with slow flow of materials. On the other hand, theobjective which the applicant set itself is the production of a milkprotein fraction comprising, among other constituents, lactoferrin andlactoperoxidase, but comprising especially a higher proportion of theother proteins than in the starting milk or whey. For that, theapplicant has developed a process with much higher flows than thosedescribed in the documents analyzed above, this process allowing a moreselective binding in relation to certain proteins.

The document WO93/13676 discloses a process which makes it possible toisolate lactoferrin and lactoperoxidase from whey by passage over acation-exchange resin at high flow. This process is distinguishable fromthe process according to the present invention in that the flow isgreater than in the process according to the invention (greater than 5m/h) and in that it aims to purify the lactoferrin and thelactoperoxidase, and not to obtain a particular milk protein fractioncontaining a relatively high level of the other proteins and endowedwith improved biological properties.

There is also known, from the document EP-704218, an agent intended forstrengthening the bones, this agent comprising a basic protein fractionor a basic peptide fraction, derived from milk and obtained by passingmilk over a cationic resin and eluting. However, the adsorption andelution conditions described in this document are distinct from thoseused in the process according to the invention and make it possible toisolate a protein fraction distinct from that obtained according to thepresent invention.

The documents JP-8165249, JP-9187250, JP-9294537 and EP-1010430 describecompositions intended for strengthening the bones and/or for preventingperiodontal diseases, these compositions comprising a milk-derived basicprotein fraction obtained by adsorption of the milk and elution on acationic resin. However, the adsorption and elution conditions describedin these documents are distinct from those used in the process accordingto the invention and make it possible to isolate a protein fractiondistinct from that obtained according to the present invention.

Thus, it is surprising that the applicant has discovered a novel processfor isolating milk proteins which make it possible to obtain a milkprotein fraction endowed with improved biological properties, inparticular a milk protein fraction promoting growth of the osteoblastsand inhibition of the proliferation of the preosteoclasts and of theosteoclasts.

The subject of the invention is a process for isolating milk proteinsfrom milk or whey comprising the following steps:

-   -   a) the milk or the whey is sterilized and defatted;    -   b) the milk fraction derived from step a) is passed over a        cation-exchange resin conditioned in an elution column;    -   c) the fraction retained on the resin is eluted with an aqueous        salt solution;    -   d) the eluate resulting from step c) is desalted, preferably by        ultrafiltration and diafiltration, and then sterilized,        preferably by microfiltration.

This process being characterized in that:

-   -   α) the cation-exchange resin is a resin grafted onto strong acid        functional groups;    -   the parameter BV denoting the ratio of the volume of raw        material to the volume of wet resin in the column,    -   the parameter SV denoting the ratio of the rate of feeding the        column to the volume of wet resin in the column,    -   the parameter LV denoting the ratio of the rate of feeding the        column to the section of the column,    -   β) during step b), the binding parameters have the following        values:        -   BV_(f) is between 50 and 400, preferably between 80 and 300;        -   SV_(f) is between 2 and 40 h⁻¹;        -   LV_(f) is greater than or equal to 1 m/h and less than or            equal to 5 m/h.    -   γ) during step c), the elution parameters have the following        values:        -   BV_(e) is between 1.5 and 7;        -   LV_(e) is less than 1 m/h, preferably less than 0.5 m/h.

It is possible to use as starting material in the process according tothe invention either milk or whey, preferably derived from cows. Whey isthe residual liquid obtained after extracting the proteins and the fatfrom milk or milk serum. Three categories of whey can be distinguished.The first two categories are classified according to the acidity of thewhey which may be less than or greater than 1.8 g of lactic acid/l:sweet whey, derived from the manufacture of cooked or uncooked pressedcheese (emmenthal, saint-paulin, etc.) and acid whey, derived fromcasein or other cheeses obtained by mixed or lactic curdling (softcheeses, fromage frais). The average composition of sweet whey is, as aguide, for 61 g of dry matter content per kg of whey, from 42 to 48 g oflactose, 8 g of proteins, 2 g of fat, 5 to 7 g of minerals, 1 to 5 g oflactic acid and the remainder as minerals and vitamins.

The ideal whey obtained by microfiltration of milk on a support havingan average porosity of 0.1 μm is known.

According to a first variant of the invention, there is used as startingmaterial milk, and advantageously cows' milk, whose composition allows,by the process according to the invention, the production of a proteinisolate having more advantageous biological properties. This variantalso makes it possible to obtain a protein yield greater than thevariants using whey as starting material.

According to a second preferred variant of the invention, acid whey fromcasein manufacture is used as starting material. This variant representsan important economic advantage since the starting material is aby-product derived from industrial exploitation and therefore of a lowcost.

In the first step of the process, the milk or whey is sterilized anddefatted by skimming, in a known manner:

The skimming of milk denotes the separation of the cream from the milk,regardless of the process used to obtain this separation.

Traditionally, the manufacture of cream is carried out according to anatural process: when the milk is allowed to stand, the elementsconstituting it separate according to their density. The fat globules,being lighter than water, rise to the surface to form a layer of cream.In industrial production, the formation of the cream is accelerated bypassing the milk into a centrifugal cream separator.

Usually, the pasteurization is carried out by controlled heating ofshort duration so as to remove the pathogenic microorganisms which maybe present in the cream. Advantageously, the pasteurization is carriedout at a temperature of between 65 and 95° C., preferably between 80 and95° C. It may be possible for example to treat the milk or the whey for15 seconds at a temperature of between 65 and 82° C. It is also possibleto sterilize the starting milk or whey by microfiltration on a filterhaving pores with a diameter ranging from 0.1 to 2 μm.

The sterile and defatted raw material is then passed over a cationexchange resin. According to the invention, the cation-exchange resin isa resin grafted by strong acid functional groups and having anion-exchange capacity of between 200 and 1000 μE/ml, preferably between400 and 700 μE/ml. The expression strong acid functional group isunderstood to mean an acid functional group having a pKa≦2. Inparticular, it may be grafted by sulfonic acid functional groups,generally in the form of sulfonate salts for their use, the nature ofthe salt determined by the solution which served to pack the columnbefore carrying out the process. Preferably, grafting by aromatic oraliphatic groups carrying sulfonic acid functional groups, still morepreferably in the form of propyl sulfonate salts, is chosen. The resinon which the sulfonic acid functional groups are grafted may be of anytype, in particular of the polyacrylic or polystyrene type. The particlesize of the resin is advantageously between 100 μm and 900 μm,preferably between 200 and 750 μm, still more preferably between 250 and600 μm. The resin which can be used according to the invention shouldpreferably have a density greater than 1.15.

Among the commercially available resins which can be used in the presentinvention, there may be mentioned in particular: the Trisacryl SP® resinmarketed by the company BIOSEPRA, the MacroPrep High S® resin marketedby the company BioRad. Preferably, a polystyrene resin grafted by alkylor aryl sulfonate functional groups is chosen.

The resin is conditioned in a column before its use, in a manner knownto the person skilled in the art, by treating with a disinfectantsolution and rinsing in order to avoid contamination by microorganisms.It is then optionally equilibrated by passing a buffer solution andrinsing.

Step b) of binding the sterile and defatted raw material, milk or whey,is carried out under the following preferential conditions:

The resin is conditioned in a column whose temperature is kept between 2and 15° C., preferably between 4 and 12° C. Preferably the column is fedwith raw material through the bottom. Advantageously, the operation iscarried out in a fluidized bed. Preferably, the binding parameters areadjusted so that one or more of the following conditions are met:

-   -   BV_(f) is between 80 and 150, preferably between 80 and 120;    -   SV_(f) is between 5 and 40 h⁻¹, preferably between 8 and 20 h⁻¹,        still more preferably between 8 and 15 h⁻¹;    -   LV_(f) is between 3 and 4.8 m/h, preferably between 3.2 and 4        m/h.

Preferably, all of the following conditions are met during step b):

-   -   BV_(f) is between 80 and 120;    -   SV_(f) is between 8 and 15 h⁻¹;    -   LV_(f) is between 3 and 4.8 m/h. during step c):    -   BV_(e) is between 3 and 7;    -   LV_(e) is less than 1 m/h.

By ion exchange, the proteins of the milk fraction used as startingmaterial come and bind to the acid functional groups of the resin. Thechoice of the binding parameters according to the invention makes itpossible to carry out a selective binding of the proteins onto thecolumn. Under conventional conditions for binding milk to a cationicresin, lactoferrin and lactoperoxidase, which are predominant,preferably bind to the resin. In the process according to the invention,the other minor, proteins are favored by the binding conditions definedabove and their proportion in this mixture of proteins bound to theresin is significantly greater than their proportion in the startingmaterial. The process according to the invention thus makes it possibleto isolate a milk fraction having a new protein composition relative tothe prior art milk protein compositions and exhibiting advantageousbiological properties.

Step c) of eluting the bound proteins is carried out under the followingpreferential conditions:

The resin is kept at a temperature of between 2 and 15° C., preferablybetween 4 and 12° C. Preferably, the column is fed with raw material(aqueous salt solution) through the top. The aqueous saline solutionused for carrying out the invention is generally a solution of achloride of an alkali metal such as K+, Na+, Ca+, Mg+. Preferably, anaqueous solution of sodium chloride is used. Advantageously, the aqueoussalt solution has a concentration of between 2 and 25%, preferably of 5to 15% by weight of salt per weight of liquid. Preferably, the ionicstrength of the aqueous salt solution is between 1 and 2 M. The pH ofthe eluting solution is generally between 6 and 7, advantageouslybetween 6.5 and 7.

Preferably, the elution parameters meet one or more of the followingconditions:

-   -   BV_(e) is between 3 and 7, and preferably between 3 and 5;    -   LV_(e) is less than 0.5 m/h.

In a known manner, the column of resin is washed before another use.

After this step, the eluate obtained, containing the mixture of milkproteins, is subjected in a known manner to one or more ultrafiltrationand diafiltration steps intended to remove the salts. Other means knownto persons skilled in the art, such as electrodialysis or passage overweak anionic and cationic resins, can be used in this step as areplacement for ultrafiltration and diafiltration. Preferably, thistreatment is carried out until a permeate having a conductivity of lessthan 15 mS is obtained. Any other process which makes it possible toremove the salts, such as in particular electrodialysis, can also beused in place of this step. The solution is then subjected tomicrofiltration intended to sterilize the ultrafiltration retentatebefore drying. Other technical means may be used to sterilize the milkfraction obtained in this step, in particular a suitable heat treatment,ultrasound or pulsed electric field.

Preferably, the desalted and sterilized product is dried in order toobtain the milk fraction derived from the process of the invention inthe form of a powder, which then allows its packaging and its storage.In a known manner, the drying may be performed by freeze-drying or byspray-drying.

The milk protein fraction, preferably derived from cows' milk andobtained by the process according to the invention is novel. In driedform, it is characterized in that it has:

-   -   a protein content of greater than 90%,    -   a mineral salt content of less than 1%,    -   a fat content of less than 1%,    -   a lactose content of less than 1%,    -   a moisture content of less than 5%,    -   a lactoferrin content of less than 80%,    -   a pH in solution at 2% of between 6 and 7.5,    -   a UV-visible spectrophotometric purity defined by an OD⁴¹²/OD²⁸⁰        ratio <0.15,    -   at least 1% of proteins having an isoelectric point greater than        or equal to 8, the percentages being given by weight relative to        the weight of dry matter content of the milk fraction according        to the invention.

In the case where the starting material used is milk and not whey, theproduct obtained by the process according to the invention ischaracterized by the additional conditions:

-   -   presence of at least 40% of proteins having an isoelectric point        greater than or equal to 8,    -   the lactoferrin content is greater than 30% and less than 80%,    -   the lactoperoxidase activity is greater than or equal to 120        ABTS units per mg of isolate (ABTS=2,2′-azino-bis-(3-ethyl Benzo        Thiazoline 6-Sulfonic Acid).

The measurement of OD at 412 nm gives a quantitative evaluation of thelactoferrin and of lactoperoxidase present in the milk protein fraction.

The measurement of OD at 280 nm gives a quantitative evaluation of allthe proteins present in the milk protein fractions.

The OD⁴¹²/OD²⁸⁰<0.15 ratio shows that the lactoferrin andlactoperoxidase are underrepresented compared with the other proteins inthe composition according to the invention in comparison with theproportions in which they are present in milk and in the milk proteinfractions of the prior art.

Preferably, the milk protein fraction obtained by the process accordingto the invention corresponds to at least one of the followingcharacteristics:

-   -   a protein content of greater than 95%,    -   a mineral salt content of less than 0.5%,    -   a fat content of less than 0.5%,    -   a lactose content of less than 0.5%,    -   a moisture content of less than 4%,    -   a pH in solution at 2% of between 6 and 7.2,    -   a UV-visible spectrophotometric purity defined by an OD⁴¹²/OD²⁸⁰        ratio <0.1,    -   contains at least 1% of proteins having an isoelectric point of        between 8.2 and 8.7.

In the case where the starting material used is milk and not whey, theproduct obtained by the process of the invention is characterized by theadditional preferred condition:

-   -   the lactoferrin content is greater than 50% and less than 80%.

The milk protein fractions according to the invention exhibitadvantageous properties: in particular they promote the growth ofosteoblast cells and that of intestinal cells.

The milk protein fractions of the invention are also effective forinhibiting the growth of osteoclasts and preosteoclasts.

These properties, and the already known properties of the milk proteinfractions of the prior art make it possible to envisage the use of thesecompositions in numerous applications, in particular in the food andpharmaceutical sectors. The subject of the invention is therefore alsoany food or pharmaceutical composition and any hygiene productcomprising a milk protein fraction according to the invention.

A food composition according to the invention may be for example adietary milk, in particular a milk intended as infant food, obtained bysimple rehydration of the powder of milk protein fraction according tothe invention. The subject of the invention is therefore the use of amilk protein fraction according to the invention for the preparation ofa dietary milk.

The subject of the invention is also food compositions comprising a milkprotein fraction according to the invention and other food ingredients.It is possible in particular to envisage adding the milk proteinfraction according to the invention to a cows' milk in order to enrichit in certain proteins. The presence of calcium in foods comprising themilk protein fraction according to the invention will be particularlybeneficial since this milk protein fraction improves the absorption ofcalcium by the human body, in particular the binding of calcium in thebone tissue.

The subject of the invention is therefore also the combination of a milkprotein fraction according to the invention with calcium.

The invention also relates to the dietary kits comprising severalconstituents packaged separately, intended for preparation of the foodimmediately before use and comprising powder of milk protein fractionaccording to the invention.

Such foods may be used for the prevention of pathologies such as: growthretardation, osteoporosis, bone fragility, bone fractures, rheumatism,osteoarthritis, periodontal diseases, intestinal barrier deficiency, andthe like.

A pharmaceutical composition according to the invention, comprising atleast one milk protein fraction according to the invention and apharmaceutically acceptable carrier may be used in the treatment of oneor more of the following pathologies: growth retardation, osteoporosis,bone fragility, bone fractures, rheumatism, osteoarthritis, periodontaldiseases, intestinal barrier deficiency, and the like.

Of course such a composition may additionally comprise one or more othertherapeutic active agents. Calcium may be advantageously combined withthe milk protein fraction according to the invention. Such a combinationforms part of the present invention. Indeed, a milk protein fractionaccording to the invention makes it possible to improve the absorptionof calcium in the body, in particular the binding of calcium in the bonetissue, which makes it possible to improve the bone strengtheningeffect.

Calcium with vitamin D may also advantageously be combined with the milkprotein fraction according to the invention. Such a combination alsoforms part of the present invention. Indeed, vitamin D improves theintestinal absorption of calcium and a milk protein fraction accordingto the invention makes it possible to improve the binding of calcium inthe bone tissue. This synergistic effect makes it possible in particularto improve bone health.

Pharmaceutical compositions or dietary supplements according to theinvention may be administered in any appropriate form such as in theform of a powder, granules, tablets, gelatin capsules, a drink, such asfor example as a solution or as a syrup. The frequency of administrationand the dose to be administered are adjusted in a known manner accordingto the weight and the age of the individual.

Also included in the present invention are the hygiene products, inparticular the products intended for oral hygiene, such as toothpastesin gel or paste form, mouthwashes, chewing gums, comprising a milkprotein fraction composition.

EXAMPLES I—Preparation of the Milk Protein Isolates

General Conditions:

The resin used in the examples below is a Trisacryl SP® resin. Itundergoes the following conditioning treatments before its initial use:bringing into contact with a 0.4% aqueous solution of the disinfectantASEPTO®, at the rate of 3 liters of disinfectant per kilo of wet resin,followed by rinsing. Equilibration by passing an acetate buffer (80 mM,pH=5.3) containing calcium chloride (33 g/l) and potassium chloride (50g/l), at the rate of 4 liters of this buffer per kilo of wet resin.Finally, the resin is rinsed with water until an eluate is obtainedwhich has a pH of between 6 and 7 and a conductivity of less than 5 mS.

After each sequence of binding of raw material and elution, the resin iscleaned by enzymatic treatment with Bacillus licheniformis alkalineprotease in buffer at pH=8, at 60° C. for 2 hours, followed by treatmentwith an NaCl solution at 100 g per liter for thirty minutes (repeatedtwice). Before its reuse it is subjected to the conditioning treatmentpresented above.

The ultrafiltration is carried out on the eluate at a temperature ofbetween 5 and 10° C. on organic membranes whose cut-off is 10 kD. Thevolume concentration factor used is between 10 and 60, so as to obtain aretentate having the highest possible dry extract, preferably between 15and 20%. The diafiltration is performed at a temperature of between 15and 25° C. on the same membranes, using a volume of demineralized waterof between 8 and 10 times the volume of retentate obtained at the end ofthe ultrafiltration until a conductivity of the permeate of less than 15mS is obtained.

The microfiltration is carried out at 35° C. using the retentateobtained from ultrafiltration/diafiltration on ceramic membranes with acut-off of 1.4 μm.

The microfiltration permeate is dried in a turbine spray-drying towerwith an inlet temperature of 140° C. and an outlet temperature of 80° C.

Characteristic of the Resin:

The SPEC 70 resin consists of a chemically inert and mechanicallyresistant support.

Chemically, the support is manufactured by polymerization of AMPS:2-acrylamide 2-methylpropanesulfonic acid. The resin has an ion-exchangecapacity of between 400 and 700 μE/ml and a protein binding capacitymeasured on lactoferrin (minimum 20 mg/ml) or lactoperoxidase (minimum40 mg/ml) or lysozyme (minimum 70 mg/ml); its density is greater than1.15 and its particle size is between 250 and 560 μm.

Example 1

300 000 liters of skimmed milk, heat-treated at 68° C. for 15 seconds,are passed at 10° C. over 3000 liters of cation-exchange resin(reference SPEC 70 supplied by the company BIOSEPRA) at a flow rate of14000 liters/h in an ascending flow in two fractionating columns inparallel and each having a radius of 1 meter.

The proteins bound to the resin are eluted with 9000 liters of a 10%sodium chloride solution in a descending flow.

The eluate obtained has a pH of 6.5 and a dry extract of 80 g/kg. It ispassed over ultrafiltration membranes with a cut-off of 10 kD and havinga surface area of 17 m², provided by DSS, at 11° C. with a permeationflow of 161/h/m² until a volume concentration factor of 7 is obtained.

The retentate has a pH of 5.9 and a dry extract of 170 g/kg; in order toremove the salt, this retentate is then diafiltered on the samemembranes at 25° C. with water in an amount of 70% of the volume ofeluate used and until a volume concentration factor of 9.5 is finallyobtained with a permeation flow of 161/h/m².

The diafiltered retentate has a pH of 6.5 and a dry extract of 95 g/kg;it is then passed over microfiltration membranes having a porosity of1.4 μm at 30° C. with a permeation flow of 320 l/h/m². The permeate ofthis microfiltration has a pH of 6.6 and a dry extract of 75 g/kg.

This permeate is then dried in a spray-drying tower provided with aturbine; the tower inlet temperature is 140° C. and the outlettemperature is 80° C.

The powdered isolate thus obtained has the following characteristics:

-   -   moisture : 4.7%,    -   proteins (N×6.38): 96.2% of which:        -   lactoferrin: 54%        -   lactoperoxidase: 125 ABTS unit/mg    -   ash: 0.1% of which:        -   Na: 0.02%        -   Cl: 0.44%    -   spectrophotometric purity: OD⁴¹²/OD²⁸⁰=0.06.

The electrophoretic profile obtained by isoelectric focusing with silvernitrate development is illustrated by FIG. 1. In FIG. 1 are compared thedistribution of the isoelectric points of the milk protein fractionaccording to the invention (noted LN06) and a reference product notedStd which is a protein calibration kit marketed by the company Pharmaciaunder the reference 17047101 and called “Isoelectric FocusingCalibration Kit Broad PI 3-10”. It is observed that proteins having highisoelectric points (around 8.4) are predominantly present. This productcomprises about 50% by weight of lactoferrin relative to the totalweight of the isolate.

Example 2

4.4 liters of native acid serum from casein manufacture (pH 4.7, dryextract 65 g/kg) are passed at 10° C. over 22 ml of cation-exchangeresin (reference SPEC 70 provided by the company BIOSEPRA) at a flowrate of 400 ml/h in descending flow in a column having a diameter of 15mm.

The proteins bound to the resin are eluted with 90 ml of a 10% sodiumchloride solution in descending flow.

In order to remove the salt, the eluate obtained (100 ml) is thendialyzed against demineralized water for 48 hours at 4° C.

The dialyzed product has the following characteristics:

-   -   dry extract: 1.5 g/kg,    -   pH: 5.4,    -   conductivity: 24 μS,    -   OD⁴¹²<0.005,    -   OD²⁸⁰=4.6 (0.46 diluted 10 times).

The electrophoretic profile makes it possible to observe that at least1% of the proteins present have an isoelectric point greater than orequal to 8. This product contains about 3% lactoferrin by weightrelative to the total weight of the protein isolate.

II—Biological Tests

A—General Methods

1. Tests of Proliferation of MC3T3 Cells (Osteoblast Cell Line)

The MC3T3 cells are cultured at 37° C. in a humid atmosphere containing5% CO₂. The culture medium used is α-MEM (alpha-Modified Eagle Medium)supplemented with 10% fetal calf serum and 50 U/ml of penicillin and 50μg/ml streptomycin.

To test the effect of the various proteins on cell growth, the cells areinoculated on 48-well plates at a density of 5×10⁴ cells/cm². 48 hoursafter inoculation, the proteins are added to the cells. The proteinsolutions are prepared in the culture medium at a concentration of 10mg/ml and then filtered on a 0.22μ filter and diluted to the desiredconcentration in the culture medium immediately before use. The cellsare counted after 72 h of culture by evaluating the quantity of DNA.

2. Tests of Proliferation of RAW 264.7 Cells (Preosteoclast Cell Line)

The RAW 264.7 cells are cultured at 37° C. in a humid atmospherecontaining 5% of CO₂. The culture medium used is DMEM (Dubelcco'sModified Eagle Medium) containing 25 mM of glucose, 4 mM glutamine and1.5 g/l of bicarbonate supplemented with 10% fetal calf serum and 50U/ml of penicillin and 50 μg/ml streptomycin.

To test the effect of the various proteins on cell growth, the cells areinoculated on 48-well plates at a density of 5×10⁴ cells/cm². 48 hoursafter inoculation, the proteins are added to the cells. The proteinsolutions are prepared in the culture medium at a concentration of 10mg/ml and then filtered on a 0.22μ filter and diluted to the desiredconcentration in the culture medium immediately before use. The cellsare counted after 72 h of culture by evaluating the quantity of DNA.

3. Test of Proliferation of Caco-2 Cells (Intestinal Cell Line

The Caco-2 cells are cultured at 37° C. in a humid atmosphere containing5% of CO₂. The culture medium used is DMEM (Dubelcco's Modified EagleMedium) containing 25 mM glucose supplemented with 15% fetal calf serum,1% of nonessential amino acids, 6 mM glutamine and 50 U/ml of penicillinand 50 μg/ml streptomycin.

To test the effect of the various proteins on cell growth, the cells areinoculated on 48-well plates at a density of 4×10⁴ cells/cm². 48 hoursafter inoculation, the proteins are added to the cells. The proteinsolutions are prepared in the culture medium at a concentration of 10mg/ml and then filtered on a 0.22μ filter and diluted to the desiredconcentration in the culture medium immediately before use. The cellsare counted after 72 h of culture by evaluating the quantity of DNA.

B—Results

The tests described above were carried out on three products:

-   -   a cows' milk protein isolate composed of 90% lactoferrin        (control product): T₁,    -   a cows' milk protein isolate whose lactoferrin content is less        than 0.01% (control product): T₂,    -   the product prepared in example 1: P₁,    -   the product prepared in example 2: P₂.

The results of these tests are presented in Table I: TABLE I InhibitionStimulation Stimulation of the of the of the prolifer- prolifer- Prod-proliferation ation of RAW ation of Caco- uct Concentration of MC3T3cells 264.7 cells 2 cells T1 1.0 mg/ml +65% −70% +67% 0.1 mg/ml +37%−30% +40% T2 1.0 mg/ml +9% −8% +9% 0.1 mg/ml +6% −4% — P1 1.0 mg/ml +36%−70% +40% 0.1 mg/ml +15% −22% +30% P2 1.0 mg/ml +31% −32% +41% 0.1 mg/ml+6% −17% +37%

The concentration denotes the concentration of milk protein isolates inthe culture medium.

The prior art tends to demonstrate that the activity of a milk proteinisolate is linked to the percentage of lactoferrin which it contains(see results of T₂).

Contrary to this preconception of the prior art, the results of thetests presented above show that milk protein isolates of the invention,although containing little lactoferrin compared with the control T₁,exhibit a remarkable activity in the tests of cell growth on osteoblastsand on intestinal cells and of inhibition of proliferation ofpreosteoclasts.

1. A process for isolating milk proteins from milk or whey comprisingthe following steps: a) the milk or the whey is sterilized and defatted;b) the milk fraction derived from step a) is passed over acation-exchange resin conditioned in an elution column; c) the fractionretained on the resin is eluted with an aqueous salt solution; d) theeluate resulting from step c) is desalted and sterilized and wherein α)the cation-exchange resin is a resin grafted onto strong acid functionalgroups; the parameter BV denoting the ratio of the volume of rawmaterial to the volume of wet resin in the column, the parameter SVdenoting the ratio of the rate of feeding the column to the volume ofwet resin in the column, the parameter LV denoting the ratio of the rateof feeding the column to the section of the column, β) during step b),the binding parameters have the following values: BV_(f) is between 5and 400; SV_(f) is between 2 and 40 h⁻¹; LV_(f) is greater than or equalto 1 m/h and less than or equal to 5 m/h. γ) during step c), the elutionparameters have the following values: BV_(e) is between 1.5 and 7;LV_(e) is less than 1 m/h.
 2. The process as claimed in claim 1, whereinthe starting material is cows' milk.
 3. The process as claimed in claim1, wherein the starting material is a casein acid whey.
 4. The processas claimed in claim 1, wherein the cation-exchange resin is a resingrafted by acid functional groups with a pKa≦2 having an ion-exchangecapacity of between 200 and 1000 μE/ml.
 5. The process as claimed inclaim 4, wherein the resin is grafted by sulfonate salt or sulfonic acidfunctional groups.
 6. The process as claimed in claim 5, wherein theresin is grafted by propyl sulfonate or propylsulfonic functionalgroups.
 7. The process as claimed in claim 1, wherein the particle sizeof the resin is between 100 μm and 900 μm.
 8. The process as claimed inclaim 1, wherein during step b) of binding of the raw material, one ormore of the following conditions are met: BV_(f) is between 80 and 150;SV_(f) is between 5 and 40 h⁻¹; LV_(f) is between 3 and 4.3 m/h.
 9. Theprocess as claimed in claim 1, wherein the following conditions are metduring step b): BV_(f) is between 80 and 120; SV_(f) is between 8 and 15h⁻¹; LV_(f) is between 3 and 4.8 m/h. during step c): BV_(e) is between3 and 7; LV_(e) is less than 1 m/h.
 10. The process as claimed in claim1, wherein during step b), the resin is conditioned in a column whosetemperature is kept between 2 and 15° C.
 11. The process as claimed inclaim 1, wherein during step c) for elution of the bound proteins, atleast one of the following conditions is met: BV_(e) is between 3 and 5;LV_(e) is less than 0.5 m/h.
 12. The process as claimed in claim 1,wherein during step c), the resin is conditioned in a column whosetemperature is kept between 2 and 15° C.
 13. The process as claimed inclaim 1, wherein the aqueous saline solution used for carrying out theinvention is a solution of a chloride of an alkali metal chosen from K+,Na+, Ca+, Mg+.
 14. The process as claimed in claim 12, wherein theaqueous saline solution is an aqueous sodium chloride solution.
 15. Theprocess as claimed in claim 14, wherein the aqueous saline solution hasa concentration of between 2 and 25% by weight of salt per weight ofliquid.
 16. The process as claimed in claim 14, wherein the aqueoussaline solution has an ionic strength of between 1 and 2 M.
 17. Theprocess as claimed in claim 1, wherein the pH of the aqueous salinesolution for elution is between 6 and
 7. 18. The process as claimed inclaim 1, wherein preceding claims, characterized in that the desaltingis carried out by ultrafiltration and diafiltration.
 19. The process asclaimed in claim 18, wherein the ultrafiltration and diafiltrationtreatments are carried out until a permeate having a conductivity ofless than 15 mS is obtained.
 20. The process as claimed in claim 1,wherein the sterilization is carried out by microfiltration.
 21. Theprocess as claimed in claim 1, wherein the desalted and sterilizedproduct is dried so as to obtain the milk fraction derived from theprocess of the invention in the form of a powder.
 22. A milk proteinfraction, obtained by the process according to claim
 1. 23. A milkprotein fraction, having the following characteristics: a proteincontent of greater than 90%, a mineral salt content of less than 1%, afat content of less than 1%, a lactose content of less than 1%, amoisture content of less than 5%, a lactoferrin content of less than80%, a pH in solution at 2% of between 6 and 7.5, a UV-visiblespectrophotometric purity defined by an OD⁴¹²/OD²⁸⁰ ratio <0.15,contains at least 1% of proteins having an isoelectric point greaterthan or equal to 8, the percentages being given by weight relative tothe weight of dry matter content of the milk fraction.
 24. The milkprotein fraction as claimed in claim 23, wherein it corresponds to atleast one of the following characteristics: a protein content of greaterthan 95%, a mineral salt content of less than 0.5%, a fat content ofless than 0.5%, a lactose content of less than 0.5%, a moisture contentof less than 4%, a lactoferrin content of less than 80%, a pH insolution at 2% of between 6 and 7.2, a UV-visible spectrophotometricpurity defined by an OD⁴¹²/OD²⁸⁰ ratio <0.1, contains at least 1% ofproteins having an isoelectric point of between 8.2 and 8.7.
 25. Themilk protein fraction as claimed in claim 23 it is derived from cows'milk.
 26. The milk protein fraction as claimed in claim 25, comprisingat least 40% of proteins having an isoelectric point greater than orequal to
 8. 27. The milk protein fraction as claimed in claim 25 havinga lactoferrin content greater than or equal to 30% and a lactoperoxidaseactivity greater than or equal to 120 ABTS units per mg of isolate. 28.The milk protein fraction as claimed in claim 22 which is derived from acasein acid whey.
 29. A combination of a milk protein fraction accordingto claim 22 with calcium.
 30. The combination as claimed in claim 29,additionally including vitamin D.
 31. A food composition, comprising amilk protein fraction according to.
 32. A dietary kit comprising apowder of milk protein fraction as claimed in claim
 22. 33. A dietarymilk prepared from a milk protein fraction as claimed in claim
 22. 34. Afood intended for the prevention of a pathology selected from: growthretardation, osteoporosis, bone fragility, bone fractures, rheumatism,osteoarthritis, periodontal diseases, and intestinal barrier deficiency,said food being prepared from a milk protein fraction as claimed inclaim
 22. 35. A food intended to promote the growth of osteoblastsand/or of intestinal cells and/or to inhibit the growth ofpreosteoclasts, said food being prepared from a milk protein fraction asclaimed in claim
 22. 36. A pharmaceutical composition, comprising atleast one milk protein fraction as claimed in and a pharmaceuticallyacceptable carrier.
 37. A medicament intended for the prevention and/ortreatment of a pathology selected from: growth retardation,osteoporosis, bone fragility, bone fractures, rheumatism,osteoarthritis, periodontal diseases, and intestinal barrier deficiency,said medicament comprising a milk protein fraction as claimed in claim22.
 38. The use of a milk protein fraction as claimed in claim 22, forthe preparation of a medicament intended to improve the absorption ofcalcium in the body.
 39. A medicament intended to promote the growth ofosteoblasts and/or of intestinal cells and/or to inhibit the growth ofpreosteoclasts, said medicament comprising a milk protein fraction asclaimed in claim
 22. 40. A hygiene product, comprising at least one milkprotein fraction as claimed in claim 22.